Acid-labile subunit (ALS) of insulin-like growth factor binding protein complex

ABSTRACT

The acid-labile sub-unit (ALS) of insulin like growth factor binding protein complex in biologically pure form is described. 
     ALS has a molecular weight between 80-115 kd as determined by SDS polyacrylamide gel electrophoresis, run under reducing conditions; and a partial N-terminal amino acid sequence as follows: 
                     Gly         AspProGlyThrProGlyGluAlaGluGlyProAlaCysProAlaAla           Cys-Ala                  
wherein the first amino acid may be Gly or Ala.
 
     Also described are methods of producing ALS, compositions containing the in-vivo IGF protein complex, methods of detecting ALS in body fluids, recombinant nucleic acid sequences encoding ALS, and expression vectors and host cells containing such nucleic acid sequences.

FIELD OF INVENTION

This invention relates to a previously unknown and uncharacterisedpolypeptide, hereinafter referred to as the acid-labile sub-unit (ALS)of insulin like growth factor (IGF) binding protein complex.

Peptides of the insulin-like growth factor (IGF) family resemble insulinboth in their structure and in many of their actions. The IGF familyconsists of two members designated IGF-I and IGF-II (IGFs). The IGFsexhibit a broad spectrum of biological activity, including anabolicinsulin-like actions (e.g. stimulation of amino acid transport andglycogen synthesis), mitogenic activity and the stimulation of celldifferentiation.

Human IGF-I and IGF-II have been extensively characterized, and havebeen found to have molecular weight of approximately 7.6 Kd (IGF-I) and7.47 Kd (IGF-II).

Unlike most peptide hormones, IGFs are found in the circulation(in-vivo) and in cell culture medium in association with one or morebinding proteins. The nature of the binding protein or binding proteinsassociated with the IGFs has been the subject of debate. Wilkins, J. R.and D'Ercole, A. J. (1985, J. Clin. Invest. 75, 1350-1358) have proposedthat the in-vivo form of IGF is a complex comprising IGF in associationwith six identical sub-units having a molecular weight of 24 Kd to 28Kd. In a second proposal, the in-vivo form of IGF is said to beassociated with an acid-stable binding protein and an acid-labileprotein(s) to generate a complex of approximately 150 Kd (Furlanetto, R.W. (1980) J. Clin. Endocrinol. Metab. 51, 12-19).

We have previously identified an acid-stable serum protein which has asingle IGF-binding site per molecule, is immunologically related to the150 Kd in-vivo form of IGF and which has an apparent molecular weight ofapproximately 53 Kd (Baxter, R. C., and Martin, J. L. (1986) J. Clin.Invest. 78, 1504-1512; and Martin, J. L. and Baxter, R. C. (1986) J.Biol. Chem. 261, 8754-8760). This 53 Kd IGF binding protein (BP53)appears to correspond to the acid stable binding protein proposed byFurlanetto. The 53 Kd protein is the highest molecular weight member ofa family of acid-stable IGF binding proteins. Other members of thisfamily have approximate molecular weights of 20, 34, 36, 30 and 47 Kd,and collectively fall within the definition “acid-stable IGF bindingprotein”.

We have now surprisingly identified an acid-labile protein, which whenincubated with the 53 Kd acid stable protein occupied by IGF converts itto a high molecular weight complex, corresponding to the in-vivo form ofIGF.

SUMMARY OF THE INVENTION

According to one aspect of the invention there is provided theacid-labile sub-unit (ALS) of insulin like growth factor binding proteincomplex in biologically pure form, which preferably has the followingpartial N-terminal amino acid sequence:

Cly AspProGlyThrProGlyGluAlaGluGlyProAlaCysProAlaAla Cys-Alawherein the first amino acid may be Gly or Ala.

In another aspect of the invention there is provided a composition ofmatter consisting essentially of the acid-labile sub-unit (ALS) of theinsulin like growth factor binding complex.

In another aspect of the invention there is provided a composition,reconstituted from three polypeptide components, namely, IGF, BP-53 andALS. The composition may be formulated to be in association with one ormore pharmaceutically acceptable carriers or excipients.

In yet another aspect of the invention there is provided a process forthe preparation of ALS, which comprises the steps of:

(a) applying a source of ALS to a support matrix having attached theretoIGF bound to or associated with the acid-stable IGF binding protein,whereby the ALS in the applied material binds to the acid stable bindingprotein and non-bound material is separated from the support matrix; and

(b) selectively eluting and recovering the ALS protein from the IGFprotein complex.

Preferably, ALS is prepared by a process comprising the steps of:

(a) binding IGF to a support matrix;

(b) adding the acid-stable IGF binding protein to the support matrixsuch that it binds to or is associated with the IGF;

(c) applying a source of ALS to the support matrix whereby the ALS inthe applied material binds to the acid stable protein and non-boundmaterial is separated from the support matrix;

(d) selectively eluting the ALS protein from the IGF protein complex;and

(e) optionally further fractionating the recovered ALS by HPLC or FPLC.

According to a further aspect of the invention there is provided amethod for detecting the levels of ALS in body fluids, which comprisesfractionating the body fluids on a size fractionation matrix to separatefree ALS from the other components of the insulin growth factor bindingcomplex, and thereafter quantitating the levels of ALS in thefractionated sample.

In still another aspect of the invention there is provided a recombinantnucleic acid sequence encoding the acid-labile sub-unit (ALS) of insulinlike growth factor. The recombinant nucleic acid sequence preferablyencodes a polypeptide having the following partial N-terminal amino acidsequence:

Gly AspProGlyThrProGlyGluAlaGluGlyProAlaCysProAlaAla Cys-Ala.wherein the first amino acid is Gly or Ala.

The invention also relates to an expression vector containing arecombinant nucleic acid sequence encoding ALS, host cells transformedwith such a vector, and ALS when produced by such host cells.

In yet another aspect of the invention there are provided polypeptidescomprising fragments of ALS, and nucleic acids comprising sequencesencoding same, which include or encode residues 1-5, 2-7, 5-9, 7-11,8-14, 11-15, 13-17, 3-9, 2-8, 4-10, 6-12, 8-14, 10-16, 12-1, 1-6, 3-9,5-11, 7-13, 9-15, 11-17, 4-9, 6-11, 8-13, 10-15, or 12-17 of ALS.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to ALS, a polypeptide which binds to, andstabilizes in-vivo, a complex between IGF and its acid-stable bindingprotein BP-53. IGF can be IGF-I or IGF-II.

BP-53 is a glycoprotein, that is, one or more carbohydrate chains areassociated with the BP-53 polypeptide sequence. Where mention is made ofthe acid-stable IGF binding protein or BP-53, it is to be understood torefer to an acid-stable protein capable of binding to insulin likegrowth factor, and capable of forming a complex with ALS and IGF. Aslong as the acid-stable IGF binding protein or BP-53 satisfies thesefunctions, it may be non-glycosylated, partly glycosylated, modified byway of amino acid deletions or substitutions or insertions, and may havea molecular weight of 20, 30, 34, 36, 47 and 53 Kd. The precisemolecular weight of this component is generally unimportant.

In accordance with the present invention and using the methods disclosedherein, said ALS is biologically pure. By biologically pure is meant acomposition comprising at least 65% by weight of ALS and preferably atleast 75% by weight. Even more preferably, the composition comprises atleast 80% ALS. Accordingly, the composition may contain homogeneous ALS.In this specification, the term “biologically pure” has the same meaningas “essentially or substantially pure”.

Where this invention relates to a composition of matter consistingessentially of ALS, the term “composition of matter” is to be consideredin a broad context. The composition of matter may be ALS itself, or ALSin association with one or more pharmaceutically or veterinariallyacceptable carriers or excipients. Suitable carriers may include water,glycerol, sucrose, buffers or other proteins such as albumin, etc. Theterm “consisting essentially of” has the same meaning as “biologicallypure” discussed above.

By binding to IGF is meant the ability of ALS to bind to complexesformed when IGF is bound or associated with an acid-stable component,BP-53.

ALS is a glycoprotein, that is, one or more carbohydrate chains areassociated with the ALS polypeptide sequence. This invention extends toALS in its fully glycosylated, partially glycosylated ornon-glycosylated forms, which may be readily prepared according tomethods well known in the art. For example, ALS prepared according tothe methods disclosed herein may be reacted with enzymes, such asendoglycosidases, to remove N-linked carbohydrate either partially ortotally. O-linked carbohydrate may similarly be removed by well knownmethods.

As mentioned previously, ALS preferably has the following partialN-terminal amino acid sequence:

-Gly AspProGlyThrProGlyGluAlaGluGlyProAlaCysProAlaAla Cys-Alawhere the first amino acid may be Gly or Ala.

It is to be understood, however, that the ALS of the present inventionis not restricted to possessing the above N-terminal amino acidsequence. Rather, ALS is functionally defined as an acid-labilepolypeptide which is capable of binding to or associating with complexesformed when IGF is bound or associated with the acid stable bindingprotein BP-53 defined above. The definition ALS extends to encompasssynthetic and naturally occurring amino acid substitutions, deletionsand/or insertions to the natural sequence of ALS, as will be readilyapparent to the skilled artisan. For example, genetic engineering meanscan be readily employed using known techniques to substitute, deleteand/or insert amino acids.

Generally, and in no way limiting the invention, ALS may becharacterized in that it:

(i) is acid-labile, that is, it is unstable at a pH less than 4,(ii) binds to an acid stable IGF binding protein which is occupied byIGF, and(iii) has an approximate molecular weight between 80 Kd and 115 Kd asdetermined by SDS-PAGE.

ALS referred to herein is human ALS. Animal ALS, which is capable offorming a complex with animal IGF, is also to be understood to fallwithin the scope of the term ALS.

ALS is contemplated herein to be useful in the preparation of thephysiological IGF complex which comprises IGF, BP-53 and ALS. Such acomplex may be useful in wound-healing and associated therapiesconcerned with re-growth of tissue, such as connective tissue, skin andbone; in promoting body growth in humans and animals; and in stimulatingother growth-related processes. The ALS protein also confers aconsiderable increase in the half-life of IGF in-vivo. The half-life ofIGF per se, unaccompanied by binding proteins, is only a few minutes.When IGF is in the form of a complex with the acid-stable IGF bindingprotein, and the ALS protein, its half-life is increased to severalhours, thus increasing the bio-availability of IGF with its attendanttherapeutic actions. Furthermore, pure ALS may be used to raise specificmonoclonal or polyclonal antibodies, in order to establish aradioimmunoassay or other assay for ALS. Measurement of ALS in humanserum may be useful in diagnosing the growth hormone status of patientswith growth disorders.

The IGF binding protein complex formed by admixing ALS, IGF and the acidstable protein BP-53, where each component is preferably in biologicallypure form, may be formulated with suitable pharmaceutically and/ortherapeutically or veterinarially acceptable carriers and used forexample, in growth promotion or wound treatment in human and non-humananimals. Examples of pharmaceutically acceptable carriers includephysiological saline solutions, serum albumin, or plasma preparations.Depending on the mode of intended administration, compositions of theIGF binding protein complex may be in the form of solid, semi-solid orliquid dosage preparations, such as for example, tablets, pills,powders, capsules, liquids, suspensions or the like. Alternatively, theIGF binding protein complex may be incorporated into a slow releaseimplant, such as osmotic pumps for the release of material over anextended time period.

The amount of the IGF binding protein complex administered to humanpatients or animals for therapeutic purposes will depend upon theparticular disorder or disease to be treated, the manner ofadministration, and the judgement of the prescribing physician orveterinarian.

ALS may be purified from human serum or plasma, or plasma fractions suchas Cohn Fraction IV. Purification from whole serum is preferred, thisbeing the most economical and plentiful source of material and givingthe highest yield. Purification of ALS exploits the physiologicalinteraction between IGF, BP-53 and ALS. ALS is recovered from humanserum by passing the serum through a support matrix having IGF-BP-53bound or associated therewith. Reference to association means anon-covalent interaction, such as electrostatic attraction orhydrophobic interactions. ALS bound to the IGF-BP-53 affinity matrix maythen be eluted by disrupting the interaction between ALS and theaffinity matrix, for example by increasing ionic strength (e.g. at least0.3M NaCl, or other equivalent salt) or conditions of alkaline pH (abovepH 8).

A source of ALS such as whole plasma or Cohn Fraction IV thereof may befractionated on an ionic resin to enrich the amount of ALS prior toapplication to the affinity matrix. A cation exchange resin ispreferred. Optionally, ALS purified by affinity chromatography issubjected to a further purification step such as HPLC or FPLC(Trademark, Pharmacia). The HPLC step may, for example, be conductedusing a reverse phase matrix, a gel permeation matrix or an ionicmatrix.

Where this invention is concerned with antibodies which are capable ofbinding to ALS, the antibodies may be monoclonal or polyclonal. Suchantibodies may be used to measure ALS levels in serum, and may form partof a diagnostic kit for testing growth related disorders. Antibodiesagainst ALS may be prepared by immunizing animals (for example; mice,rats, goats, rabbits, horses, sheep or even man) with purified ALSaccording to conventional procedures (Goding, J. W. (1986) MonoclonalAntibodies: Principles and Practices, 2nd Edition, Academic Press).Serum proteins may, for example, be attached to a support matrix, andincubated with anti-ALS antibodies which may be labelled with reportergroups (for example, fluorescent groups, enzymes or colloidal groups) todetect ALS. Alternatively, non-labelled anti-ALS antibodies bound to ALSmay be reacted with suitable agents (such as antibodies directed againstanti-ALS antibodies or anti-immunoglobulin antibodies) to detectantibody binding, and thus quantitate ALS levels.

Where this invention relates to a recombinant nucleic acid molecule,said molecule is defined herein to be DNA or RNA, encoding ALS or partsthereof. In one embodiment, the recombinant nucleic acid molecule iscomplementary DNA (cDNA) encoding mammalian and preferably, human ALS,or parts thereof including any base deletion, insertion or substitutionor any other alteration with respect to nucleotide sequence or chemicalcomposition (e.g. methylation). ALS encoded by cDNA is herein referredto as recombinant ALS.

A recombinant nucleic acid which exhibits at least 60% sequence homologyor more preferably 80 to 99% homology with nucleic acid (cDNA, DNA, RNA)encoding ALS, or which encodes a protein having the biological activityof ALS, is to be regarded as nucleic acid encoding ALS.

Methods considered useful in obtaining recombinant ALS cDNA arecontained in Maniatis et. al., 1982, in Molecular Cloning: A LaboratoryManual, Cold Spring Harbour Laboratory, New York. pp 1-545. Briefly,polyadenylated mRNA is obtained from an appropriate cell or tissuesource, such as liver. Optionally, mRNA is fractionated on agarose gels,or gradient centrifugation, and translated and assayed for ALS, such as,for example, by immunoprecipitation. Enriched or unenriched mRNA is usedas a template for cDNA synthesis. Libraries of cDNA clones areconstructed in the Pst1 site of a vector such as pBR 322 (usinghomopolymeric tailing) or other vectors; or are constructed by ligatinglinkers (such as Eco R1 linkers) onto the ends of cDNA, which is thencloned into a vector having sites complementary to said linkers.Specific cDNA molecules in a vector in a library are then selected usingspecific oligonucleotides based on the aforementioned N-terminal aminoacid sequence of ALS. Alternatively, commercially available human lambdalibraries may be screened with oligonucleotides. In an alternativeapproach, the cDNA may be inserted into an expression vector such aslambda gt 11, with selection based on the reaction of expressed proteinwith a specific antibody raised against purified ALS. In any event, onceidentified, cDNA molecules encoding all or part of ALS are then ligatedinto expression vectors. Additional genetic manipulation is routinelycarried out to maximise expression of the cDNA in the particular hostemployed.

Accordingly, ALS is synthesized in vivo by inserting said cDNA sequenceinto an expression vector, transforming the resulting recombinantmolecule into a suitable host and then culturing or growing thetransformed host under conditions requisite for the synthesis of themolecule. The recombinant molecule defined herein should comprise anucleic acid sequence encoding a desired polypeptide inserted downstreamof a promoter functional in the desired host, a eukaryotic orprokaryotic replicon and a selectable marker such as one resistant to anantibiotic. The recombinant molecule may also require a signal sequenceto facilitate transport of the synthesized polypeptide to theextracellular environment. Alternatively, the polypeptide may beretrieved by first lysing the host cell by a variety of techniques suchas sonication, pressure disintegration or detergent treatment. Hostscontemplated in accordance with the present invention can be selectedfrom the group comprising prokaryotes (e.g., Escherichia coli, Bacillussp., Pseudomonas sp.) and eukaryotes (e.g., mammalian cells, yeast andfungal cultures, insect cells and plant cultures). The skilled personwill also recognize that a given amino acid sequence can undergodeletions, substitutions and additions of nucleotides or tripletnucleotides (codons). Such variations are all considered within thescope of the present invention. Additionally, depending on the hostexpressing recombinant ALS, said ALS may or may not be glycosylated.Generally, eukaryotic cells, for example mammalian cells and the like,will glycosylate the recombinant ALS. Prokaryotic cells, for example,bacteria such as Escherichia coli and the like, will not glycosylate therecombinant ALS. Both glycosylated and non-glycosylated ALS areencompassed by the present invention, as has been previously mentioned.

Abbreviations

-   IGF—insulin-like growth factor-   SDS-PAGE—sodium dodecylsulphate polyacrylamide gel electrophoresis-   Kd or K—kilodaltons-   GH—growth hormone

The following drawings and Examples are illustrative of, but in no waylimiting, on the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Affinity chromatography of ALS. A 132-ml pool of fractionspartially purified by DEAE-Sephadex chromatography was loaded at 0.13ml/min onto a 1×15 cm affinity column containing a mixture of IGF-I andIGF-II covalently bound to agarose, to which BP-53 had beennoncovalently adsorbed. The column was washed with 150 ml of 50 mM Naphosphate, pH 6.5 (Wash #1) and 50 ml of 5 mM Na phosphate, 50 mM NaCl,pH 6.5 (wash #2), at 1 ml/min. ALS was eluted by applying 50 mMTris-HCl, 0.3 M NaCl, pH 8.5, at 0.5 ml/min.

FIG. 2: SDS-polyacrylamide gel electrophoresis of purified ALS. Leftpanel: Untreated, acidified, and N-glycanase-treated samples (15μg/lane) run under nonreducing conditions. Right panel: the same samplesin reverse order, run under reducing conditions. Gels were stained withCoomassie blue. The molecular masses (in kDa) of standard proteins,shown in the right hand lane for the reduced gel, are also indicated byarrows on the left for standards run on the nonreduced gel.

FIG. 3 shows fractionation of human serum on a column of DEAE-SephadexA-50. One milliliter dialyzed serum was loaded onto a 1×17.5 cm. gelbed, the column was washed with 35 mL 0.05 mol/L Tris-HCl. pH 8.2 andelution commenced with 50 mL of the same buffer containing 0.15 mol/LNaCl. Elution was then continued with the same buffer containing 0.6mol/L NaCl. Fractions of 1 mL were collected and assayed for absorbanceat 280 nm and BP-53 by RIA. ALS was determined on 20-μL aliquots of peakB fractions by the routine assay method;

FIG. 4 depicts the generation of the 150K complex fromDEAE-Sephadex-fractionated serum. Peak A and B pools, as in FIG. 3, wereprepared by DEAE-Sephadex chromatography of 10 mL serum, thenfractionated by Superose-12 chromatography. The samples, injected in avolume of 200 μL each, were peak A (a: 100 μL), peak B (b: 100 μL) peaksA and B (c: 100 μl each), mixed and incubated for 1 h at 22° C. beforeloading, and whole serum (d: 33 μL). BP-53 immunoreactivity was measuredon 50 μL of each 0.5 mL fraction. Arrows indicate 150K, 60K and 35Kmarkers;

FIG. 5 shows the comparison of BP-53 immunoreactivity and ALS activity,as indicated in serum fractionated on Superose-12. Each fraction was 0.5mL. The arrows indicate 150K, 60K and 35K markers. Note that the methodused to detect ALS protein only detects protein not present as the 150Kcomplex;

FIG. 6 depicts acid lability of ALS activity. Samples of normal serum(a) or partially pure ALS (b) were diluted in ALS assay buffer andadjusted to the pH values shown with 1 mol/L HCl or NaOH. After 30 minat 22° C., the samples were reneutralized and assayed for ALS activityin the routine assay (10 μL serum or 600 ng ALS preparation/incubation);

FIG. 7 shows the effect of IGFs on ALS binding to BP-53. Left panel:Increasing concentrations of BP-53 were incubated in a 300 μl reactionvolume with [¹²⁵I]-labeled ALS tracer in the presence or absence ofIGF-I or IGF-II (50 ng/tube), as indicated. Right panel: Competitivebinding study in which 10 ng BP-53 plus 10 ng IGF-I or IGF-II wasincubated in 300 μl with ALS tracer and increasing concentrations ofunlabelled ALS. Tracer bound to BP-53 was immunoprecipitated withanti-BP-53 antiserum R-7.

FIG. 8 shows the effect of ALS on IGF binding to BP-53. Left panel:Increasing concentrations of BP-53 were incubated in a 300 μl reactionvolume with [¹²⁵I]-labeled IGF-I or IGF-II tracer (IGF-I* or IGF-II*) inthe presence or absence of ALS (100 ng/tube), as indicated. Right panel:Competitive binding study in which 0.25 ng BP-53, in the presence orabsence of 100 ng ALS, was incubated in 300 μl with IGF-II tracer andincreasing concentrations of unlabelled IGF-I or IGF-II, as indicated.Tracer bound to BP-53 was immunoprecipitated with anti-BP-53 antiserumR-7.

FIG. 9 shows the effect of BP-53 and ALS on the gel chromatographicprofile of [¹²⁵I]-labeled IGF-II tracer. Samples of 200 μl containing50,000 cpm of IGF-II tracer, preincubated 2 h at 22° C. in the presenceor absence of BP-53 (1 ng/200 μl) or ALS (100 ng/200 μl), werechromatographed on a Superose-12 high performance chromatography columnin 50 mM Na phosphate, 0.15 M NaCl, 0.02% Na azide, 0.1% bovine albumin,pH 6.5. Fractions of 0.5 ml were collected at 1 ml/min, and theradioactivity in each fraction determined. On each panel the threearrows indicate, from left to right, molecular weight markers of 150kDa, 60 kDa and 7.5 kDa. Left panel: solid symbols, IGF-II tracer; opensymbols, tracer plus ALS. Right panel: solid symbols, tracer plus BP-53;open symbols, tracer plus BP-53 plus ALS.

FIG. 10 shows competition by increasing concentrations ofacidified-neutralized human serum from normal, hypopituitary oracromegalic subjects in the routine ALS assay, in which 600 ng ofpartially purified ALS/250 μL incubation medium (i.e. 2.4 μg/mL) gave a150K/60K ratio of approximately 1 in the absence of added serum. Theserum concentration is expressed in terms of volume (a) or in terms ofthe immunoreactive BP-53 content (b). The acidified-neutralized serumsamples illustrated contained 4.49 μg/mL (normal), 0.93 μg/mL(hypopituitary), or 10.49 μg/mL (acromegalic) BP 53 by RIA; and

FIG. 11 depicts competition by pure BP-53 in the routine ALS assay, (a)the effect of increasing BP-53 concentrations after preincubationwithout IGFs or with a 3.5-fold molar excess of pure human IGF-I orIGF-II, as indicated. Panel (b), shows the effect of a fixed BP-53concentration (0.8 μg/mL) preincubated with increasing concentrations ofIGF-I or IGF-II.

EXAMPLES Example 1 Materials

Fresh human serum for ALS preparation was obtained from laboratoryvolunteers. Cohn Fraction IV of human plasma, provided by CommonwealthSerum Laboratories, Melbourne, Australia, was used as starting materialto prepare human IGF-I and IGF-II, and the IGF-binding protein BP-53.DEAE-Sephadex A-50, SP-Sephadex C-25, electrophoresis standards, and theSuperose 12 HR 10/30 column were obtained from Pharmacia, Sydney;Affi-Gel 10 and Affi-Gel 15 were purchased from Bio-Rad; and the PolyWAXLP (polyethleneimine) anion exchange HPLC column (200×4.6 mm) was fromPolyLC, Columbia, Md. All other reagents were at least analytical grade.

Human IGF-I and IGF-II were isolated and iodinated as previouslydescribed (Baxter, R. C., and De Mellow, J. S. M. (1986) Clin.Endocrinol. 24, 267-278; and Baxter, R. C., and Brown, A. S. (1982)Clin. Chem. 28, 485), and IGF-I tracer was further purified byhydrophobic interaction chromatography (Baxter, R. C., and Brown, A. S.(1982) Clin. Chem. 28, 485). The 53K IGF-binding protein BP-53 waspurified from Cohn fraction IV as previously described (Martin, J. L.,and Baxter, R. C. (1986) J. Biol. Chem. 261, 8754-8760), and a covalentcomplex with [¹²⁵I]IGF-I, cross-linked using disuccinimidyl suberate,was prepared and purified by gel chromatography according to the methodof Baxter, R. C., and Martin, J. L. (1986) J. clin. Invest. 78,1504-1512. A 28K IGF-binding protein BP-28 was purified from humanamniotic fluid by affinity chromatography and reverse phase highpressure liquid chromatograph according to Baxter, R. C., Martin, J. L.and Wood, M. H. (1987), J. Clin. Endocrinol. Metab. 65, 423-431.

ALS Iodination and Radioimmunoassay:

[¹²⁵I]-labeled ALS was prepared by reacting 5 μg ALS in 50 μl M Naphosphate buffer, pH 7.4, for 20 sec with 1 mCi Na ¹²⁵I and 10 μgchloramine-T, then terminating the reaction with 50 μg Na metabisulfite.An antiserum against ALS was raised by immunizing a rabbit over a 7-weekperiod with 4 doses of approximately 100 μg purified ALS.Radioimmunoassay incubations in 0.5 ml final volume contained antiserumat 1:50,000 final dilution, [¹²⁵I]-labeled ALS (approx. 10,000 cpm pertube), and ALS in the range 0.5-100 ng/tube. After a 16 h incubation at22° C., bound and free tracer were separated by centrifugation followingthe addition of goat anti-rabbit immunoglobulin (2 μl), carrier normalrabbit serum (0.5 μl), and, after 30 min, 1 ml 6% polythylene glycol in0.15 M NaCl.

Assay for ALS:

The routine assay for ALS activity was developed, based on theconversion of a covalent BP-53-IGF-I complex of approximately 60K to150K form in the presence of ALS.

Samples to be tested for ALS activity were diluted to 200 μL in buffercontaining 50 mmol/L sodium phosphate, 0.15 mol/L NaCl, and 0.2 g/Lsodium azide, pH 6.5, with 10 g/L bovine albumin. Cross-linkedBP-53-IGF-I tracer (−80,000 cpm; 4 ng) was added in 50 μL of the samebuffer. After 25-30 min. of incubation at 22° C., 200 μL of the mixturewas applied using a V-7 injector valve (Pharmacia) to a Superose-12 gelpermeation column eluting at 1.0 mL/min (−2 megapascal pressure) inassay buffer without albumin. The column was calibrated with rabbitimmunoglobulin G (Pentex; −150K), which eluted mainly in fractions22-24, peaking in fraction 23; BP-53-IGF-I tracer (−60K), which elutedmainly in fractions 25-27, peaking in fraction 26; IGF-I tracer bound toBP-28 (−35K, peaking in fraction 28); and IGF-I tracer (7.5K, peaking infraction 33). Plotted as log (molecular mass) vs. elution volume, thesefour markers yielded a linear calibration curve (not shown). As aquantitative index of ALS activity (i.e. the degree of conversion ofBP-53 to the 150K complex), the total radioactivity in fractions 22-24was divided by that in fractions 25-27 to give a 150K/60K ratio. Thevalues of this ratio typically varied between 0.1 and 2.0. Thecoefficient of variation of the 150K/60K ratio, based on analysis ofvariance of eight duplicate measurements covering a wide range ofvalues, was 3.2%. Because each chromatography run took 30 min., and theprecision of the assay was high, each determination was generallyperformed singly within each experiment.

In the absence of ALS, the radioactivity was found predominantly infractions 25-27, corresponding to a molecular mass of 60K, typicallygiving a 150K/60K ratio of 0.10 or lower. Increasing concentrations ofALS in the preincubation caused increasing conversion of the 60K tracerto the 150K form (fractions 22-24), giving higher values for the150K/60K ratio. Both IGF-I and IGF-II tracers, preincubated with pureBP-53 but not covalently cross-linked, could also be converted to 150Kby incubation with ALS. Other IGF acid-stable binding proteinsstructurally related to BP-53 but of smaller size (such as those of 20,24, 26, 30 and 47K) also participate in this reaction to formcorresponding smaller complexes. Cross-linked BP-53-IGF-II tracer wasnot tested. A dose-response curve using the purified ALS preparationproduced according to Example 2 was constructed using cross-linkedBP-53-IGF-I tracer. A highly reproducible sigmoidal semilog plot wasobtained, which could be used as a standard curve for quantitating theALS in unknown samples (for example during purification). A similarresult is obtained if the tracer complexed to ALS is precipitated withan anti-ALS antiserum instead of being fractionated on a Superose-12column.

Example 2 Purification of ALS

Fresh human serum or Cohn Factor IV paste of human plasma were used as asource of ALS. Fresh human serum (100-130 ml) was dialyzed against 2×50vol of 0.05 M Tris-HCl pH 8.2 at 2° C., then loaded onto a column ofDEAE Sephadex A-50 (5×23 cm) equilibrated with dialysis buffer at 22° C.The column was washed with 2 liters of dialysis buffer, then with 2-2.5liters of the same buffer containing 0.15 M NaCl. This step removed allof the immunoreactive BP-53 from the column. ALS was eluted by applying1 liter of 0.05 M Tris-HCl, 0.6 M NaCl, pH 8.2, pumping at 1 ml/min.Fractions of 10 ml were collected and assayed for ALS activity andabsorbance at 280 nm. Active fractions were combined (approximately 140ml total) and dialyzed at 2° C. against 5 liters of 50 mM sodiumphosphate, 0.02% Na azide, pH 6.5.

Where Cohn Factor IV is the source of ALS, the frozen paste (600 g) wasbroken into small pieces and extracted for 16 h at 2° C. by stirringwith 3 liters 50 mM Tris-HCl, 0.15 M NaCl, 0.02% sodium azide, pH 8.2.The mixture was centrifuged 30 min at 12000 rpm in the GSA rotor of aSorvall RC5C centrifuge, yielding a turbid green-brown supernatantfraction (2.8 liter). This was divided into two equal portions andloaded by gravity feed onto two columns of DEAE Sephadex A-50 (5×22 cm)equilibrated with extraction buffer, and each column was washed with 2liters of buffer. At this stage a predominant blue-green band wasconcentrated in the upper half of the column. Sometimes this bandstarted to migrate through the column during the washing step; in thesecases the washing volume was decreased to 1 liter. ALS was eluted fromthe column by a linear 0.15-0.35 M NaCl gradient in 50 mM Tris-HCl,0.02% sodium azide, pH 8.2 (2 liter total volume). Fractions of 10 mlwere collected and assayed for ALS activity and absorbance at 280 nm (orprotein by a Biuret method). Active fractions from the two parallelcolumns were combined (approximately 1 liter total), diluted two-foldwith 50 mM sodium phosphate pH 6.5, and the pH adjusted to 6.5 by slowaddition of 1 M HCl. Since the active fractions corresponded closelywith the blue-green protein in the eluted fractions, this provided aconvenient visual marker for the progress of the activity through theion-exchange procedure.

The ALS containing fractions obtained from plasma or Cohn Factor IV, asdetailed above, were applied to either one of two IGF affinity columns:(1) Affi-Gel 15 column (1×12 cm) to which 3 mg IGF-II had been coupledexactly as previously described (Martin, J. L., and Baxter, R. C. (1986)J. Biol. Chem. 261, 8754-8760), or (2) Affi-Gel 10 column (1×15 cm) towhich a mixture containing approximately 5 mg IGF-1 and 2 mg IGF-II hadbeen coupled by the same procedure. The affinity column was loaded withBP-53, prepared exactly as previously described (Martin, J. L., andBaxter, R. C. (1986) Supra). Briefly, 600 g Cohn paste was homogenizedwith 5 vol of 2 M acetic acid, 75 mM NaCl, the mixture was centrifuged,and the supernatant was depleted of endogenous IGFs by stirring 2-3 dayswith approximately 400 ml packed volume of SP-Sephadex C-25 which hadbeen equilibrated in the homogenizing buffer at pH 3.0. The mixture wascentrifuged to remove the gel, and the supernatant was adjusted to pH6.5 in two steps, as previously described (Martin, J. L., and Baxter, R.C. (1986) Supra). The pH 6.5 supernatant was then pumped atapproximately 0.5 ml/min onto the affinity column, and the column waswashed at 1-2 ml/min with 250 ml of 50 mM Na phosphate, 0.5 M NaCl, pH6.5, and 100 ml of 50 mM Na phosphate, pH 6.5.

ALS containing fractions from DEAE-Sephadex chromatography were pumpedat 0.1-0.15 ml/min onto the IGF affinity column loaded with BP-53. Thistypically resulted in the retention of over 90% of the ALS activity. Thecolumn was washed at 1 ml/min with 150 ml of 50 mM Na phosphate, pH 6.5,and 50 ml mM NaCl, 5 mM Na phosphate pH 6.5, to lower the bufferingcapacity of the column. ALS was eluted by applying 50 mM Tris-HCl, 0.3 MNaCl, pH 8.5 to the column at 0.5 ml/min. This is illustrated in FIG. 1which shows a plot of elution volume from the affinity column againstALS (μg/ml) and absorbance at 280 nm. Fractions of 2 ml were collectedin siliconized glass tubes and assayed for ALS activity.

SDS-PAGE (10%) of the immunopurified ALS, under reducing conditions,yielded a doublet of closely associated bands with an approximatemolecular weight of 90K. The doublet may be due to varying glycosylationof ALS. No other bands were present, this indicating that the ALS washomogeneous.

As an optional final purification step affinity-purified ALS wasfractionated by high-performance anion exchange chromatography. Sampleloads of 0.5 ml per run were applied to a PolyWAX high performance anionexchange column equilibrated at 1.5 ml/min in 0.05 M ammonium hydrogencarbonate (unadjusted pH=7.8). The ALS was eluted by applying a linearsalt gradient (Model 680 Gradient Controller, Waters, Milford, Mass.)from 0.05 M to 0.5 M ammonium hydrogen carbonate (pH adjusted) over 15min at 1.5 ml/min. In some preparations a concave gradient was used(gradient #7, Model 680 Gradient Controller) over the same concentrationrange, with comparable results. Absorbance at 280 nm was monitored usinga Waters Model 441 Absorbance Detector. Fractions of 0.75 ml werecollected and assayed for ALS activity. A single major protein peakemerged from the column after 9-10 min elution at 1.5 ml/min when alinear gradient was employed, or 11-12 min using a concave gradient. Allof the detectable ALS activity, determined by RIA, was associated withthis peak, with the recovery of applied activity estimated at over 75%,and a further increase in specific activity of 1.6-fold. The ALSactivity was always associated with a single peak.

FIG. 2 shows purified ALS after HPLC fractionation electrophoresed on alinear 10-15% polyacrylamide gel, under both reducing and non-reducingconditions. The preparation appeared as a doublet of apparent molecularmass 84 and 86 KDa under either non-reducing (left panel) or reducingconditions. Acidification of the protein (prepared by adjusting 35 μg ofALS in 50 μl 0.05M ammonium hydrogen carbonate to pH 3 with 20 μl of 1Macetic acid, incubating 15 min. at 22° C., and neutralizing with 10 μlof 2M Tris base), which results in a substantial loss of activity, hadno effect on the protein's mobility on SDS-PAGE when run eithernon-reduced or reduced. However, treatment with N-glycanase (25 μg ALSboiled in 40 μl 0.5% SDS for 3 min., then diluted in 0.55 M Naphosphate, pH 8.6 and Nonidet P-40 to final concentrations of 0.2 M and1.25% respectively; then N-glycanase (Genzyme Corp., Boston, Mass.) wasadded to a final concentration of 60 units/ml, and the mixture incubatedat 16 h at 27° C.) to remove N-linked carbohydrate resulted in asignificant decrease in apparent molecular mass, to 80 kDA non-reduced(left panel) and 66 kDA (right panel). Notably, the protein migrated asa single band after deglycosylation with N-glyoanase, suggesting thatthe doublet seen in the native preparation is due to at least twoglycosylation variants. Under reducing conditions, the deglycosylatedpreparation showed several bands in the range 50-60 kDa, suggesting thatfurther deglycosylation might be possible.

Table 1 summarizes the results of a typical ALS purification, one offour performed on a similar scale and with similar results. Fractionseluted from the DEAE-Sephadex column by 0.05 M Tris-HCl, 0.6 M NaCl, pH8.2 (DEAE eluate #2), contained over 60% of the applied ALSimmunoreactivity and 13% of the total protein, whereas fractions elutedwith buffer containing 0.15 M NaCl (DEAE eluate #1) contained only 15%of the ALS activity, but 79% of the protein. Further purification ofDEAE eluate #2 fractions by affinity chromatography on a column of BP-53non-covalently bound to agarose-IGF yielded a 200-fold increase in ALSspecific activity.

The purification strategy employed was constrained by the fact that thesub-unit is irreversibly inactivated at low pH, but took advantage ofthe fact that it is reversibly dissociated from the BP-IGF complex athigh pH. The key step in the purification is an unusual application ofaffinity chromatography in which the affinity ligand (BP-53) is notattached to the agarose matrix by a covalent bond, but appears to act asa non-covalent bridge between agarose-IGF beads and the ALS. Inretrospect it is clear that the use of a covalent agarose-BP-53 matrixwould not have worked, since BP-53 unoccupied IGF-I or IGF-II is unableto bind ALS. The optional final step, high performance chromatography ona PolyWAX (weak anion-exchange) column with salt gradient elution,essentially reiterates the initial step of DEAE-Sephadex chromatography,but at much higher resolution.

Example 3 Amino-Terminal Sequence of ALS

The N-terminal sequence of ALS was determined on an estimated 35 μlsample of HPLC purified material by Edman degradation using an AppliedBiosystems 470A automatic gas-phase protein sequencer coupled to a 120APTH Analyzer using a standard PTH program. Cys residues were confirmedon a second sample after reduction with mercaptoethanol andcarboxymethylation with iodoacetic acid.

In two determinations, amino-terminal analysis showed approximatelyequimolar amounts of Gly and Ala for the first residue, despite the factthat the preparation analyzed was from the serum of a single donor.Analysis of the first 18 residues yielded the sequenceGly(Ala)-Asp-Pro-Gly-Thr-Pro-Gly-Glu-Ala-Glu-Gly-Pro-Ala-Cys-Pro-Ala-Ala-Cys-,the Cys residues in positions 14 and 18 being confirmed on a reduced andcarboxymethylated sample. This amino acid sequence shows no obvioushomology to other IGF proteins or receptors.

Example 4 DEAE-Sephadex Fractionation of Serum

The starting material was an ammonium sulfate fraction of serum from30-50% saturation prepared according to previously published tables(Green, A. A., and Hughes, W. L., Methods of Enzymol. 1, 67). Theresulting precipitate, dialyzed against an excess of 50 mmol/L Tris-HCl,pH 8.2, contained approximately 75% of the BP-53 immunoreactivity ofwhole serum. In subsequent studies, the ammonium sulfate fractionationwas found to be unnecessary, and whole serum dialyzed against Tris-HClbuffer was used. A 1×17.5 cm. column of DEAE-Sephadex A-50, equilibratedin 50 mmol/L Tris-HCl, pH 8.2, was loaded with a 1-mL dialyzed sampleand eluted with 35 mL starting buffer, 50 mL starting buffer plus 0.15mol/L NaCl, and 50 mL starting buffer plus 0.6 mol/L NaCl. In a largerscale protocol, 10-mL dialyzed samples were loaded onto a 1.5×20 cmcolumn and eluted with 50, 100 and 100 mL, respectively, of the threebuffers. The major protein peak eluting in the presence of 0.15 mol/LNaCl was termed peak A, and the peak emerging in 0.6 mol/L NaCl wastermed peak B (FIG. 3).

The majority of immunoreactive BP-53 was found in the first peak (peakA), whereas the second peak (peak B) contained ALS activity with verylittle BP-53 immunoreactivity (FIG. 3 bottom). A small amount of ALSactivity also was detected in fractions corresponding to the descendingside of peak A (not shown). Similar results were obtained in sixseparate experiments.

FIG. 4, representative of three similar experiments, shows the BP-53immunoreactivity profiles for these protein peaks, separately and afterpreincubation together when fractionated by Superose-12 chromatography.The BP-53 immunoreactivity from peak A eluted primarily in a broad peakbetween fractions 25 and 30, corresponding to a molecular mass range ofapproximately 30-60K, with a small peak in fractions 22-24;corresponding to 150K (FIG. 4 a). The barely detectable BP-53 activityfrom peak B eluted from Superose-12 predominantly in fractions 23-25(FIG. 4 b). After mixing peaks A and B and preincubating for 60 min at22° C., over 50% of the peak A BP-53 activity had shifted from 30-60K to150K, with the remainder still at 30-60K (FIG. 4 c). This may becompared with the BP-53 profile in whole serum, in which over 90% of theactivity was at 150K and only 5-10% in the 30-60K region (FIG. 4 d). TheALS activity of peak B, as depicted in FIG. 4 c, was unaffected bydialysis of peak B fractions against Tris buffer containing no NaCl or0.6 mol/L NaCl, indicating that neither high salt nor any otherdialyzable molecule was involved in the reaction between BP-53 and theALS in peak B.

Superose-12 Fractionation of Serum:

Serum from normal subjects was diluted 1:1 with 50 mmol/L sodiumphosphate, 0.15 mol/L NaCl, and 0.2 g/L sodium azide, pH 6.5, and 200 μLwas applied to the Superose-12 column and eluted as described for theroutine ALS assay. Each fraction was then tested for BP-53 and ALSactivity.

As shown previously in FIG. 4 d, BP-53 immunoreactivity peaked infraction 23, corresponding to 150K (FIG. 5). In contrast, in threeexperiments the peak ALS activity reproducibly eluted in fraction 24(FIG. 5), corresponding to 90-110K, suggesting that there is an excessof ALS over BP-53 in serum and that the free sub-unit has an apparentmolecular mass of 90-110K. A similar peak of ALS activity was found inserum from which more than 99% of immunoreactive BP-53 (i.e. essentiallyall of the 150K complex) had been removed by affinity chromatography ona column of anti-BP#-53 antibody coupled to agarose (not shown),confirming that the ALS detectable at 90-110K was not complexed toBP-53. A comparable result was found when serum was fractionated by ionexchange chromatography, as shown in FIG. 3 and peak B was subjected toSuperose-12 chromatography.

Increasing volumes of serum when tested in the routine ALS assay, gave adose dependent increase in the 150K/60K ratio (not shown). The ALSdetectable in whole serum appeared to be GH dependent, as higheractivity was found in serum from five acromegalic subjects and loweractivity in serum from five GH-deficient subjects than was detectable insamples from normal subjects. This GH-dependence provides the basis fora diagnostic assay for determining GH levels in serum, and may beexploited in the diagnosis of growth disorders using, for example,antibodies directed against ALS.

Example 5 Acid Lability of ALS

The acid lability of purified ALS or ALS in whole serum (followingprocedure of Example 2) was evident by its irreversible inactivation onexposure to low pH. The protein appeared quite stable at pH values aslow as 5, but below this it rapidly lost activity (FIG. 7); and the150K/60K ratio decreased by over 80% at pH 3. This decrease in the150K/60K ratio is equivalent to a decrease in apparent ALS activity ofover 99%. In contrast, exposure at high pH values (up to pH 10) had noeffect on ALS activity in whole serum or the purified preparation.

Example 6 Functional Studies

To determine the binding kinetics of ALS to BP-53, incubations were setup containing [¹²⁵I]-labeled ALS and various concentrations of BP-53 andIGF-I or IGF-II. Complexes of ALS tracer with BP-53 were detected afterimmunoprecipitation using an antiserum against BP-53 which haspreviously been shown to react with the BP in both free and complexedforms (Baxter, R. C. and Martin, J. L. (1986) J. Clin. Invest. 78,1504-1512). FIG. 7 (left) shows the effect of increasing BP-53concentrations, over the range 0.25 to 100 ng/tube (0.016 to 6.3 nM), oncomplex formation. In the absence of IGF-I or IGF-II, there was littleor no reaction between ALS tracer and BP-53. In the presence of a molarexcess of IGF-I or IGF-II (50 ng/tube or 22 nM), a dose-dependentincrease in ALS tracer binding was seen, increasing to 50% specificbinding to 100 ng/tube of BP-53. Higher concentrations of BP-53 couldnot be tested due to limitations of the immunoprecipitation system.Complex formation was consistently higher in the presence of IGF-I thanIGF-II.

The binding affinity between ALS and BP-IGF complexes was estimated fromcompetitive binding studies. As shown for a typical experiment in FIG. 7(right), binding of [¹²⁵I]-labeled ALS was again greater in the presenceof IGF-I than IGF-II. In three similar experiments, the mean specificbinding (±SEM) to 10 ng/tube BP-53 (i.e. corrected for radioactivityprecipitated in the absence of BP-53) was 24.3±4.4% in the presence ofexcess IGF-I, and 19.6±3.9% in the presence of excess IGF-II (P 0.009 bypaired t-test). Increasing concentrations of unlabelled ALS caused adose-dependent displacement of [¹²⁵I]-labeled ALS fromimmunoprecipitatable complexes. Analysis of binding data by Scatchardplot revealed a nonspecific binding component (association constant <10⁶M⁻¹) and a single specific binding component with a slightly higheraffinity for BP-IGF-I than BP-IGF-II. In three similar experiments themean association constant (±SEM) for ALS binding to BP-IGF-I was6.06±0.71×10⁸ M⁻¹, and for ALS binding to BP-IGF-II, 4.12±0.29×10⁸ M⁻¹.The binding site concentration was 1.28±0.46 mol ALS/mol BP-53 in thepresent of IGF-I, and 1.18±0.29 mol/mol in the presence of IGF-II,assuming the molecular masses of ALS and BP-53 are 86 kDa and 53 kDarespectively. If the calculation is based on the reduced molecular massof 43 kDa for BP-53, the binding site concentrations are 1.04±0.37mol/mol and 0.96±0.33 mol/mol respectively. This result is consistentwith a single binding site for ALS per molecule of BP-53.

The lack of effect of ALS on the interaction between BP-53 and the IGFsis shown in FIG. 8. [¹²⁵I]-labeled IGF-II consistently showed higherbinding to increasing concentrations of BP-53 than [¹²⁵I]-labeled IGF-I.The binding of either tracer was unaffected by the addition of 100 ngpure ALS per tube (FIG. 8, left). Competitive binding curves for thedisplacement of [¹²⁵I]-labeled IGF-II from BP-53 by increasingconcentrations of unlabelled IGF-I and IGF-II are shown in FIG. 8(right). IGF-II was consistently more potent than IGF-I in displacingtracer from BP-53, and neither displacement curve was affected by theaddition of 100 ng/tube of ALS. Similar results were seen when[¹²⁵I]-labeled IGF-I was used as tracer (not shown).

To confirm that pure ALS was capable of converting the BP-53 to the 150kDa form, incubation mixtures similar to those used in the competitivebinding experiments shown in FIG. 8 were fractionated by gelchromatography on Superose 12. [¹²⁵I]-labeled IGF-II appeared as asingle peak of radioactivity, peaking in Fraction 34. Incubation of thistracer with pure ALS (100 ng/200 μl) before fractionation had no effecton the radioactive profile, indicating that ALS alone was unable to bindIGF-II tracer (FIG. 9, left). Incubation of IGF-II tracer with 1 ng/200μl pure BP-53 resulted in the conversion of 70% of the radioactivity toa 60 kDa form, i.e. BP-53-IGF-I. When this incubation also included 100ng/200 μl pure ALS, the 60 kDa complex was substantially converted to a150 kDa form (FIG. 4, right), demonstrating that complex formationrequired no components other than pure IGF, pure BP-53, and pure ALS.

Example 7 Inhibition of ALS Binding to BP-53-IGF-I

Various substances were tested for their ability to inhibit tracerBP-53-IGF-I binding to ALS. Human serum, when acidified andreneutralized to destroy its endogenous ALS activity and leave itsacid-stable BP-53 intact, contained potent competing activity. Oncomparing samples from normal, acromegalic, and GH-deficient subjects inthis way in three separate experiments, the competing activity showedstrong GH-dependence, as expected for the endogenous BP-53 in suchsamples. This is illustrated for one such experiment in FIG. 10 a. Whenthe curves in FIG. 8 a were replotted in terms of the immunoreactiveBP-53 content of each sample, they became superimposable (FIG. 10 b),indicating that the endogenous BP-53 in acidified whole serum couldcompete with cross-linked tracer in the ALS reaction. Under theconditions used in this assay, approximately 1 μg BP-53/mL reactionvolume (i.e. 250 ng/250 μL) fully displaced cross-linked tracer from theBP-ALS complex, with half-maximal displacement at 200-250 ng/mL BP-53.

In contrast to the endogenous BP-53 in acidified serum, pure BP-53, whentested at up to 0.8 μg/mL, was unable to compete with cross-linkedtracer in the ALS reaction (FIG. 11). However, after preincubation for30 min at 22° C. with a 3.5-fold molar excess of pure human IGF-I orIGF-II (i.e. 500 ng IGF/μg BP-53), purified BP-53 could fully displacecross-linked tracer from the BP-53-ALS complex. Also tested, and foundnot to compete in the ALS reaction, were the following; purifiedamniotic fluid BP-28 (0.8 μg/mL), BP-28 preincubated with excess IGF-Ior IGF-II (0.5 μg/mL), or human GH (20 μg/mL). These experiments againindicate that only BP-53 that is occupied by IGF-I or IGF-II can takepart in the reaction with ALS and strongly suggest that BP-28, whetheroccupied or not, is unable to react with ALS.

The scientific articles previously referred to are incorporated hereinin their entirety.

The claims form part of the description.

TABLE 1 Purification of the acid-labile subunit from human serum TotalTotal ALS specific Purification Protein ALS Activity Purification StepVolume ml mg μg μg/mg protein Factor fold Recovery % Serum 120 118608290 0.70 1.0 100 DEAE 2530 9360 1280 0.14 0.2 15.4 eluate #1 DEAE 1321530 5060 3.31 4.7 61.0 eluate #2 Affinity 4.5 2.61 1850 709 1010 22.3eluate HPLC pool 15.75 1.22 1400 1148 1640 16.9 The purification stepswere as described in Example 2. ALS, determined by radioimmunoassay, isexpressed in terms of a pure standard preparation. DEAE eluate #1 refersto the pool of fractions eluted from DEAE-Sephadex by buffer containing0.15 M NaCl. DEAE eluate #2 refers to the pool of fractions eluted bybuffer containing 0.5 M NaCl; this pool was dialyzed before assay.Affinity eluate is the pool of fractions eluted from the affinitycolumn, then concentrated by ultrafiltration. HPLC pool is the pool ofactive fractions recovered from the HPLC step.

1. The acid-labile sub-unit (ALS) of insulin like growth factor bindingprotein complex in biologically pure form.
 2. A composition of matterconsisting essentially of the acid-labile sub-unit (ALS) of the insulinlike growth factor binding complex.
 3. ALS according to claim 1 or 2which has a molecular weight of approximately 80-115 kilodaltons asdetermined by reducing SDS-polyacrylamide gel electrophoresis.
 4. ALSaccording to any one of claims 1 to 3 having the following partialN-terminal amino acid sequence: GlyAspProGlyThrProGlyGluAlaGluGlyProAlaCysProAlaAla Cys-Ala

wherein said first amino acid may be Gly or Ala.
 5. A method for thepreparation of the in-vivo IGF binding protein complex comprising thestep of admixing together IGF, the acid-stable IGF binding protein (BP53) and ALS.
 6. A method according to claim 5, wherein the IGF,acid-stable IGF binding protein (BP 53) and ALS are biologically pure.7. A composition containing the in-vivo IGF protein complex, preparedaccording to claim 5 or 6, in association with a pharmaceutically orveterinarially acceptable carrier or excipient.
 8. A method for thetreatment of wounds comprising applying to the wound or the areaadjacent the wound, or administering to the wounded patient, atherapeutically effective amount of a composition as claimed in claim 7.9. A method for promoting cellular growth in animals and humanscomprising administering to a patient a therapeutically effective amountof a composition as claimed in claim
 7. 10. A method for the preparationof ALS which comprises the steps of: (a) applying a source of ALS to asupport matrix having attached thereto IGF bound to or associated withthe acid-stable IGF binding protein, whereby the ALS in the appliedmaterial binds to the acid stable binding protein and non-bound materialis separated from the support matrix; and (b) selectively eluting andrecovering the ALS protein from the IGF protein complex.
 11. A methodfor the preparation of ALS which comprises the steps of: (a) binding IGFto a support matrix; (b) adding the acid-stable IGF binding protein tothe support matrix such that it binds to the IGF; (c) applying a sourceof ALS to the support matrix whereby the ALS in the applied materialbinds to the acid stable binding protein and non-bound material isseparated from the support matrix; and (d) selectively eluting the ALSprotein from the IGF protein complex.
 12. A method according to claim 10or 11, wherein the source of ALS is human plasma or Cohn Fraction IV ofhuman plasma.
 13. A method according to claim 10 or 11, wherein thesource ALS applied to the support matrix is partially purified.
 14. Amethod according to claim 13, wherein the source of ALS is partiallypurified by fractionation on an ionic resin.
 15. A method according toclaim 14, wherein the ionic resin is a cation exchange resin.
 16. Amethod according to any one of claims 11 to 15, wherein the eluted ALSfrom step (d) is further fractionated by HPLC or FPLC.
 17. ALS whenprepared according to the method of any one of claims 10 to
 16. 18. Amethod for detecting ALS in a biological fluid comprising the steps of:(a) binding proteins of a biological fluid to a support matrix; (b)reacting the support matrix with antibodies directed against ALS; and(c) detecting antibody binding.
 19. A method as claimed in claim 18,wherein the biological fluid is serum.
 20. A method for detecting thelevels of ALS in a body fluid, which comprises fractionating the bodyfluid on a size fractionation matrix to separate free ALS from the othercomponents of the insulin growth factor binding complex, and thereafterquantitating the levels of ALS in the fractionated sample.
 21. A methodaccording to claim 20, wherein the levels of ALS in a body sample aredetected by measuring the extent of formation of a reconstituted insulinlike growth factor binding protein complex.
 22. A method according toclaim 20 wherein levels of ALS are quantitated by determining the extentof binding of anti-ALS antibodies to ALS in the fractionated sample. 23.A recombinant nucleic acid sequence encoding the acid-labile sub-unit(ALS) of insulin like growth factor.
 24. A recombinant nucleic acidsequence according to claim 23, which encodes ALS, said ALS having thefollowing partial N-terminal amino acid sequence: GlyAlaProGlyThrProGlyGluAlaGluGlyProAlaCysProAlaAla Cys-Ala

wherein the first amino acid may be Gly or Ala.
 25. An expression vectorcontaining the DNA sequence of claim
 23. 26. A host cell, which is aprocaryotic or eukaryotic cell, transformed with the expression vectorof claim
 25. 27. The recombinant nucleic acid sequence of claim 23 whichis cDNA.
 28. ALS when produced by the host cell of claim
 26. 29. Anantibody reagent capable of binding to ALS.
 30. An antibody reagentaccording to claim 29 which is a monoclonal or polyclonal antibody. 31.An antibody reagent according to claim 29 or 30 which is labelled withone or more reporter groups.
 32. An antibody reagent according to claim31, wherein the reporter group is selected from fluorescent groups,enzymes or colloidal groups.
 33. A nucleic acid isolate comprisingnucleic acid encoding the sequence: GlyAspProGlyThrProGlyGluAlaGlyGlyProAlaCysProAlaAla Cys-Ala

wherein the first amino acid may be Gly or Ala.
 34. A nucleic acidisolate comprising nucleic acid encoding residues 1-5, 2-7, 5-9, 7-11,8-14, 11-15, 13-17, 3-9, 2-8, 4-10, 6-12, 8-14, 10-16, 12-18, 1-6, 3-9,5-11, 7-13, 9-15, 11-17, 4-9, 6-11, 8-13, 10-15, or 12-17 of ALS. 35.The nucleic acid isolate of claim 33 or 34 further comprising areplicable vector.
 36. The nucleic acid isolate of claim 33 furthercomprising nucleic acid encoding a secretion signal sequence ligated tothe 5′ end of nucleic acid encoding the sequence: GlyAspProGlyThrProGlyGluAlaGluGlyProAlaCysProAlaAla Cys-Ala

wherein the first amino acid may be Gly or Ala.
 37. A host celltransformed with the isolate of any one of claims 33 to
 36. 38. Apolypeptide comprising a fragment of ALS comprising a sequence ofresidues 1-5, 2-7, 5-9, 7-11, 8-14, 11-15, 13-17, 3-9, 2-8, 4-10, 6-12,8-14, 10-16, 12-18, 1-6, 3-9, 5-11, 7-13, 9-15, 11-17, 4-9, 6-11, 8-13,10-15, or 12-17 of ALS.